Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiling after induction of WTX in HEK293 cells

ABSTRACT: WTX encodes a tumor suppressor, frequently inactivated in Wilms tumor, with both plasma membrane and nuclear localization. WTX has been implicated in beta-catenin turnover, but its effect on nuclear proteins is unknown. We report an interaction between WTX and p53, derived from the unexpected observation of WTX, p53 and E1B 55K colocalization within the characteristic cytoplasmic body of adenovirus-transformed kidney cells. In other cells without adenovirus expression, the C-terminal domain of WTX binds to the DNA binding domain of p53, enhances its binding to CBP, and increases CBP/p300-mediated acetylation of p53 at Lys 382. WTX knockdown accelerates CBP/p300 protein turnover and attenuates this modification of p53. In p53-reconstitution experiments, cell cycle arrest, apoptosis, and p53-target gene expression are suppressed by depletion of WTX. Together, these results suggest that WTX modulates p53 function, in part through regulation of its activator CBP/p300. Affymetrix microarray gene expression profiling was performed to identify differentially expressed genes following overexpression of WTX in HEK293 clones after 12 hour induction of WTX

ORGANISM(S): Homo sapiens

SUBMITTER: Woo Kim

PROVIDER: E-GEOD-34715 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We have previously shown that adenoviral expression of peroxisome proliferator-activated receptors (PPARs) leads to rapid establishment of transcriptionally active complexes and activation of target gene expression within 5-8 h following transduction. Here we have used the adenoviral delivery system combined with expression array analysis to identify novel putative PPARgamma target genes in murine fibroblasts and to determine the role of the A/B-domain in PPARgamma mediated transactivation of genomic target genes. Of the 257 genes found to be induced by PPARgamma2 expression, only 25 displayed A/B-domain dependency, i.e. significantly reduced induction in the cells expressing the truncated PPARgamma lacking the A/B-domain (PPARgammaCDE). Nine of the 25 A/B-domain dependent genes were involved in lipid storage and in line with this, triglyceride accumulation was considerably decreased in the cells expressing PPARgammaCDE compared to cells expressing full length PPARgamma2. Using chromatin immunoprecipitation (ChIP) we demonstrate that PPARgamma binding to genomic target sites and recruitment of the mediator component TRAP220/MED1/PBP/DRIP205 is not affected by the deletion of the A/B-domain. By contrast, the PPARgamma-mediated CBP and p300 recruitment to A/B-domain dependent target genes is abolished by deletion of the A/B-domain. These results indicate that the A/B-domain of PPARgamma2 is specifically involved in the recruitment or stabilization of CBP and p300 containing co-factor complexes to a subset of target genes. Experiment Overall Design: Total RNA samples were obtained from three individual experiments in which NIH-CAR (i.e. a clonal cell line of NIH-3T3 fibroblasts stably expressing the coxsackie-adenovirus receptor (CARÎ1)) cells were transduced with adenovirus containing empty vector (AdEmpty) followed by addition of vehicle (DMSO) or transduced with adenovirus encoding either full length HA-tagged PPARg2 or A/B-domain deleted HA-PPARgCDE and treated with rosiglitazone.

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Gene expression profiling after induction of WTX in HEK293 cells

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